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The parental-specific expression of the insulin-like growth factor-2 (Igf-2) and H19 genes was studied in rat fibroblast cells derived from a 3 day-old first-generation hybrid animal obtained by crossing Fisher and Wistar strains (F x W cells). Results showed that the reciprocal imprinting of the Igf-2 and H19 genes was conserved in the rat tissues and in the derived F x W cells when cultured with frequent transfer. Igf-2 and H19 gene expression was coordinately up-regulated upon reaching confluence, but Igf-2 RNA levels were further increased in a time-dependent manner and the repressed stale of the maternal Igf-2 allele was progressively relaxed in cultures held in the confluent state and in the presence of low serum for more than 3 days. The active expression and relaxed imprinting status of the Igf-2 gene persisted over cell generations when the growth-constraining conditions were released by trypsinization and dilution. On the contrary, the imprinting of the H19 gene appeared to be unaffected by changes in growth conditions and its expression was down-regulated when the confluent cells were passaged. Methylation of the H19 promoter and Igf-2 coding regions was increased in the F x W cells extensively held under confluence and in the derived 'post-confluent' cultures. The heritable changes in the expression. and imprinting status of the Igf-2 and H19 genes observed in the F x W cells closely resembles events described in human embryonal cancers and cancer-predisposing syndromes. The occurrence of imprinting relaxation under strong growth-inhibitory conditions supports the hypothesis that it is an epigenetic change.

The insulin-like growth factor II (IGF-II) gene is parentally imprinted in the mouse and human species. By following the inheritance of natural polymorphisms of IGF-II mRNA, we demonstrated that the tissue-specific parental imprinting of the IGF-II gene is conserved in the rat. The expression of the paternal IGF-II allele exceeded by more than 3 orders of magnitude that of the maternal allele in livers of 3-day-old Wistar x Fisher interstrain rat crosses. In contrast, the two alleles were both expressed in the rat central nervous system, which is also the only district of the organism where this gene is active in adult rodents. We also analyzed the allelic usage of the three IGF-II promoters, which generate alternatively spliced transcripts, and showed that parental imprinting of all transcription starts sites is coordinately regulated since P1, P2, and P3 are all repressed on the maternal allele in neonatal rat liver, and all of them are activated on both alleles in the choroid plexus of the central nervous system. RNase protection assays demonstrated that the activity ratio of the three IGF-II promoters can be different in tissues that show the same imprinting mode.

Post-transcriptional mechanisms operate in regulation of gene expression in bacteria, the amount of a given gene product being also dependent on the inactivation rate of its own message. Moreover, segmental differences in mRNA stability of polycistronic transcripts may be responsible for differential expression of genes clustered in operons. Given the absence of 5' to 3' exoribonucleolytic activities in prokaryotes, both endoribonucleases and 3' to 5' exoribonucleases are involved in chemical decay of mRNA. As the 3' to 5' exoribonucleolytic activities are readily blocked by stem-loop structures which are usual at the 3' ends of bacterial messages, the rate of decay is primarily determined by the rate of the first endonucleolytic cleavage within the transcripts, after which the resulting mRNA intermediates are degraded by the 3' to 5' exoribonucleases. Consequently, the stability of a given transcript is determined by the accessibility of suitable target sites to endonucleolytic activities. A considerable number of bacterial messages decay with a net 5' to 3' directionality. Two different alternative models have been proposed to explain such a finding, the first invoking the presence of functional coupling between degradation and the movement of the ribosomes along the transcripts, the second one implying the existence of a 5' to 3' processive '5' binding nuclease'. The different systems by which these two current models of mRNA decay have been tested will be presented with particular emphasis on polycistronic transcripts.

Pro-inflammatory cytokines, environmental stresses,as well as receptor tyrosine kinases regulate the activity of JNK. In turn, JNK phosphorylates Jun members of the AP-1 family of transcription factors, thereby controlling processes as different as cell growth, differentiation, and apoptosis. Still, very few targets of the JNKJun pathway have been identified. Here we show that JNK is required for the induction of c-myc expression by PDGF. Furthermore, we identify a phylogenetically conserved AP-1-responsive element in the promoter of the c-myc proto-oncogene that recruits in vivo the c-Jun and JunD AP-1 family members and controls the PDGF-dependent transactivation of the c-myc promoter. These findings suggest the existence of a novel biochemical route linking tyrosine kinase receptors, such as those for PDGF, and c-myc expression through JNK activation of AP-1 transcription factors. They also provide a novel potential mechanism by which both JNK and Jun proteins may exert either their proliferative or apoptotic potential by stimulating the expression of the c-myc proto-oncogene.

In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the his IE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, L-histidinol:NAD+ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxyl-terminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps.

Previous genetic analysis showed that the polar effects of mutations in the hisG cistron of Salmonella typhimurium are dependent on the presence of a single putative transcription termination element within the hisG gene. In fact, all proximal mutations causing translation termination are strongly polar, whereas distal ones are not. The element was mapped by isolating mutations able to relieve the polar phenotype, and they were found to be small deletions in the region downstream of the translational stop codon (M. S. Ciampi and J. R. Roth, Genetics 118:193-202, 1988). In this study, we analyzed the his-specific RNAs synthesized in vivo in different strains harboring the polar frameshift hisG2148 mutation. The nature of the polarity effects is clearly transcriptional, since shorter RNA molecules were produced. When the hisG2148 mutation was transferred in a rho background or in strains harboring the small distal deletions, an increase in readthrough transcription was observed. The transcriptional termination element was characterized in more detail by performing high-resolution S1 nuclease mapping experiments. This analysis showed that (i) termination or exonucleolytic degradation following termination produced transcripts with heterogeneous 3' ends; (ii) this process is dependent on the transcription termination factor Rho, since relief of termination occurs in a rho background; and (iii) the element appears to function as a transcription terminator, at least to some extent, even in the course of active translation of the hisG cistron.

We have studied a very unusual strong polar mutant in the intercistronic barrier between the second (hisD) and third (hisC) cistrons of the histidine operon of Salmonella typhimurium to obtain further insights into the molecular mechanisms leading to transcription termination within cistrons. We have performed a detailed transcriptional analysis in vivo and have found that the his mRNA in this polar mutant is reduced in size as a result of premature termination of transcription at a cryptic Rho-dependent site within the proximal region of the hisC cistron.

Insulin-like growth factor II (IGF-II) is a mitogenic polypeptide present in rat plasma at high levels during fetal and early postnatal life and is believed to play an important, although as yet undefined, role in fetal development. Both in humans and rats, expression of the IGF-II gene results in the appearance of several mRNA species. In the present study, cDNA and synthetic oligonucleotide probes were used to isolate and characterize the rat IGF-II gene from genomic libraries. The rat IGF-II gene extends over 12 kilobase pairs and contains two 5'-noncoding exons and three protein-coding exons. The two 5' exons represent alternative 5' regions of different mRNA molecules and are expressed from two distinct promoters. The two promoters are transcribed with different efficiencies but exhibit similar tissue-specific expression and regulation with developmental age.

Experimental animal models of bacterial meningitis are useful to study the host-pathogen interactions occurring at the cerebral level and to analyze the pathogenetic mechanisms behind this life-threatening disease. In this study, we have developed a mouse model of meningococcal meningitis based on the intracisternal inoculation of bacteria. Experiments were performed with mouse-passaged serogroup C Neisseria meningitidis. Survival and clinical parameters of infected mice and microbiological and histological analysis of the brain demonstrated the establishment of meningitis with features comparable to those of the disease in humans. When using low bacterial inocula, meningococcal replication in the brain was very efficient, with a 1,000-fold increase of viable counts in 18 h. Meningococci were also found in the blood, spleens, and livers of infected mice, and bacterial loads in different organs were dependent on the infectious dose. As glutamate uptake from the host has been implicated in meningococcal virulence, mice were infected intracisternally with an isogenic strain deficient in the ABC-type l -glutamate transporter GltT. Noticeably, the mutant was attenuated in virulence in mixed infections, indicating that wild-type bacteria outcompeted the GltT-deficient meningococci. The data show that the GltT transporter plays a role in meningitis and concomitant systemic infection, suggesting that meningococci may use l -glutamate as a nutrient source and as a precursor to synthesize the antioxidant glutathione.

PATZ is a transcriptional repressor affecting the basal activity of different promoters, whereas RNF4 is a transcriptional activator. The association of PATZ with RNF4 switches the activation to repression of selected basal promoters. Because RNF4 interacts also with the androgen receptor (AR) functioning as a coactivator and, in turn, RNF4 associates with PATZ, we investigated whether PATZ functions as an AR coregulator. We demonstrate that PATZ does not influence directly the AR response but acts as an AR corepressor in the presence of RNF4. Such repression is not dependent on histone deacetylases. A mutant RNF4 that does not bind PATZ but enhances AR-dependent transcription is not influenced by PATZ, demonstrating that the repression by PATZ occurs only upon binding to RNF4. We also demonstrate that RNF4, AR, and PATZ belong to the same complex in vivo also in the presence of androgen, suggesting that repression is not mediated by the displacement of RNF4 from AR. Finally, we show that the repression of endogenous PATZ expression by antisense expression plasmids in LNCaP cells results in a stronger androgen response. Our findings demonstrate that PATZ is a novel AR coregulator that acts by modulating the effect of a coactivator. This could represent a novel and more general mechanism to finely tune the androgen response.

The expression of the linked but reciprocally imprinted Igf2 and H19 genes is activated in adult liver in the course of tumor development. By in situ hybridization analysis we have shown that both the Igf2 and H19 RNAs are expressed in the majority of the neoplastic nodules, and that hepatocellular carcinomas are developed in an experimental model of liver carcinogenesis. H19 is also highly activated in smaller and less distinct hyperplastic regions. The few neoplastic areas showing Igf2 but no H19 RNA display loss of the maternally inherited allele at the Igf2/H19 locus. These data are compatible with the existence of a common activation mechanism of these two genes during liver carcinogenesis and with a stronger H19 induction in the pre-neoplastic lesions. By using mice carrying a deletion of the H19 endodermal enhancer, we show that this regulatory element is necessary for the activation of the Igf2 and H19 genes upon induction of liver carcinogenesis. Furthermore, multiple sites of the H19 endodermal enhancer region become hypersensitive to DNase I when the carcinogenesis process is induced. Lastly, liver tumors developed in mice paternally inheriting the H19 enhancer deletion are found to have marked growth delays, increased frequency of apoptotic nuclei, and lack of Igf2 mRNA expression, thus indicating that this regulatory element plays a major role in the progression of liver carcinogenesis, since it is required for the activation of the anti-apoptotic Igf2 gene.

RINGWe have isolated a new human RING-finger gene finger family show a variety of functions and are mostly (RNF4) that encodes a 190-amino-acid protein. RNF4, involved in the regulation of development and cell difin addition to the carboxyl-terminally located RING- ferentiation (Saurin et al., 1996). Some viral RINGfinger motif, contains two putative nuclear localiza- finger proteins are involved in transactivation of viral tion signals and stretches of acidic amino acids that or cellular genes and are essential for viral pathogenicare similar to the activation domains of some tran- ity (Saurin et al., 1996). Most of the human RINGscription factors. RNF4 was expressed at low levels in finger proteins have been localized in the nucleus or in all human tissues examined, with the notable excep- tion of very high expression in the testis. The mouse both the cytoplasm and the nucleus and are involved homolog of RNF4 was abundantly expressed in embry- in signal transduction and in oncogenesis (Saurin et onic tissues from the earliest days postgestation and al., 1996). The RING-finger motif of TRAF2 is required exhibited a ubiquitous pattern of expression as as- for the formation of the TRAF2/TANK complex, which sessed by in situ hybridization.We havemapped RNF4 in turn mediates NF-kB activation upon ligand binding to 4p16.3, a chromosome region associated with sev- to CD40 or type II tumor necrosis factor receptor eral genetic and neoplastic diseases. RNF4 spans 47 (Cheng and Baltimore, 1996). Several proteins conkb, is composed of eight exons, and maps immediately taining RING-finger motifs participate in oncogenesis proximal to the anonymous locus D4S183, between the through different mechanisms. Chromosomal translohuntingtin (HD) and the fibroblast growth factor re- cations in different neoplasias generate chimeric proceptor 3 (FGFR3) genes.

In nonpolarized cells, the small GTPase Rab5a is localized to the plasma membrane, clathrin-coated vesicles, and early endosomes. Rab5a is required for early endosome fusion in vitro and regulates transport between the plasma membrane and early endosomes, in vivo. In polarized epithelial cells endocytosis occurs from separate apical and basolateral plasma membrane domains. Internalized molecules are initially delivered to distinct apical or basolateral early endosomes. In vitro, apical early endosomes can readily fuse with one another but not with the basolateral endosomes and vice versa, thereby indicating that the apical and basolateral early endocytic pathways are controlled by distinct machineries. Here, we have investigated the localization and function of Rab5a in polarized epithelial cells. Confocal immunofluorescence microscopy on mouse kidney sections revealed association of the protein with the apical and basolateral plasma membrane domains and underlying structures. In polarized Madin-Darby canine kidney I cells, endogenous and overexpressed Rab5a have the same distribution. Moreover, overexpression of the protein causes a 2-fold increase in fluid-phase uptake from both domains of the cell, thus showing that Rab5a functions in apical and basolateral endocytosis. Our data indicate that the apical and basolateral endocytic machineries of epithelial cells share common regulatory components and that Rab5a per se is not sufficient to target endocytic vesicles to apical or basolateral early endosomes.

Insulin-like growth factor II (IGF-II) is a mitogen for many cell types and an important modulator of muscle growth and differentiation. IGF-II gene is prevalently expressed during prenatal development and its gene activity is regulated by genomic imprinting, in that the allele inherited from the father is active and the allele inherited from the mother is inactive in most normal tissues. IGF-II expression is activated in several types of human neoplasms and an alteration of IGF-II imprinting has been described in Beckwith-Wiedemann syndrome and Wilms' tumour. Here we show that monoallelic expression of IGF-II gene is conserved in normal adult muscle tissue whereas two or more copies of active IGF-II alleles, arising by either relaxation of imprinting or duplication of the active allele, are found in 9 out of 11 (82%) rhabdomyo-sarcomas retaining heterozygosity at 11p15, regardless of the histological subtype. Since IGF-II has been indicated as an autocrine growth factor for rhabdomyosarcoma cells, these findings strongly suggest that acquisition of a double dosage of active IGF-II gene is an important step for the initiation or progression of rhabdomyosarcoma tumorigenesis. Among different types of muscle tumors, relaxation of imprinting seems to arise prevalently in rhabdomyosarcomas, since we have detected only one case of partial reactivation of the maternal IGF-II allele out of 7 lelomyosarcomas tested. © 1994 Oxford University Press.

Summary We have characterized a major processed species of mRNA in the his operon of Salmonella typhimurium. In vivo and in vitro analyses of the his transcripts from wild-type and mutant strains using S1 nuclease protection assays, measurements of RNA stability, deletion mapping, gel retardation, and in vitro translation assays demonstrate that the distal portion of the polycistronic his mRNA is processed, resulting in increased stability. The processing event requires an upstream cis-acting element and translation of the cistron immediately downstream of the 5′ end of the processed species. The cistrons contained in this segment are also independently transcribed from an internal promoter which is maximally active in the absence of readthrough transcription from the primary promoter.

We have identified a human gene encoding a novel MBD2-interacting protein (MBDin) that contains an N-terminal GTP-binding site, a putative nuclear export signal (NES), and a C-terminal acidic region. MBDin cDNA was isolated through a two-hybrid interaction screening using the methyl-CpG-binding protein MBD2 as bait. The presence of the C-terminal 46-amino-acid region of MBD2 and both the presence of the acidic C-terminal 128-amino-acid region and the integrity of the GTP-binding site of MBDin were required for the interaction. Interaction between MBD2 and MBDin in mammalian cells was confirmed by immunoprecipitation experiments. Fluorescence imaging experiments demonstrated that MBDin mainly localizes in the cytoplasm but accumulates in the nucleus upon disruption of the NES or treatment with leptomycin B, an inhibitor of NES-mediated transport. We also found that MBDin partially colocalizes with MBD2 at foci of heavily methylated satellite DNA. An MBD2 deletion mutant lacking the C-terminal region maintained its subnuclear localization but failed to recruit MBDin at hypermethylated foci. Functional analyses demonstrated that MBDin relieves MBD2-mediated transcriptional repression both when Gal4 chimeric constructs and when in vitro-methylated promoter-reporter plasmids were used in transcriptional assays. Southern blotting and bisulfite analysis showed that transcriptional reactivation occurred without changes of the promoter methylation pattern. Our findings suggest the existence of factors that could be targeted on methylated DNA by methyl-CpG-binding proteins reactivating transcription even prior to demethylation.

Neisseria meningitidis strains belonging to the hypervirulent lineage ET-37 and several unrelated strains are extremely UV sensitive. The phenotype is consequent to the presence of a nonfunctional recB ET-37 allele carrying multiple missense mutations. Phenotypic analysis has been performed with congenic meningococcal strains harboring either the wild-type recB allele or the recB ET-37 allele. Congenic recB ET-37 meningococci, in addition to being sensitive to UV, were defective both in repair of DNA lesions induced by UV treatment and, partially, in recombination-mediated transformation. Consistently, the wild-type, but not the recB ET-37 , allele was able to complement the Escherichia coli recB21 mutation to UV resistance and proficiency in recombination. recB ET-37 meningococci did not exhibit higher frequencies of spontaneous mutation to rifampin resistance than recB -proficient strains. However, mutation rates were enhanced following UV treatment, a phenomenon not observed in the recB -proficient counterpart. Interestingly, the results of PCR-based assays demonstrated that the presence of the recB ET-37 allele considerably increased the frequency of recombination at the pilin loci. The main conclusion that can be drawn is that the presence of the defective recB ET-37 allele in N. meningitidis isolates causes an increase in genetic diversity, due to an ineffective RecBCD-dependent DNA repair and recombination pathway, and an increase in pilin antigenic variation.

Rab7 is a small GTPase that controls transport to endocytic degradative compartments. Here we report the identification of a novel 45 kDa protein that specifically binds Rab7GTP at its C-terminus. This protein contains a domain comprising two coiled-coil regions typical of myosin-like proteins and is found mainly in the cytosol. We named it RILP (Rab-interacting lysosomal protein) since it can be recruited efficiently on late endosomal and lysosomal membranes by Rab7GTP. RILP-C33 (a truncated form of the protein lacking the N-terminal half) strongly inhibits epidermal growth factor and low-density lipoprotein degradation, and causes dispersion of lysosomes similarly to Rab7 dominant-negative mutants. More importantly, expression of RILP reverses/prevents the effects of Rab7 dominant-negative mutants. All these data are consistent with a model in which RILP represents a downstream effector for Rab7 and both proteins act together in the regulation of late endocytic traffic.

Beckwith-Wiedeman syndrome (BWS) and Klippel-Trenaunay-Weber syndrome (KTWS) are different human disorders characterized, among other features, by tissue overgrowth. Deregulation of one or more imprinted genes located at chromosome 11p15.5, of which insulin-like growth factor 2 (IGF2) is the most likely candidate, is believed to cause BWS, whereas the etiology of KTWS is completely obscure. We report a case of BWS and a case of KTWS in a single family. The probands, sons of two sisters, showed relaxation of the maternal IGF2 imprinting, although they inherited different 11p15.5 alleles from their mothers and did not show any chromosome rearrangement. The patient with BWS also displayed hypomethylation at KvDMR1, a maternally methylated CpG island within an intron of the KvLQT1 gene. The unaffected brother of the BWS proband shared the same maternal and paternal 11p15.5 haplotype with his brother, but the KvDMR1 locus was normally methylated. Methylation of the H19 gene was normal in both the BWS and KTWS probands. Linkage between the insulin-like growth factor 2 receptor (IGF2R) gene and the tissue overgrowth was also excluded. These results raise the possibility that a defective modifier or regulatory gene unlinked to 11p15.5 caused a spectrum of epigenetic alterations in the germ line or early development of both cousins, ranging from the relaxation of IGF2 imprinting in the KTWS proband to disruption of both the imprinted expression of IGF2 and the imprinted methylation of KvDMR1 in the BWS proband. Analysis of these data also indicates that loss of IGF2 imprinting is not necessarily linked to alteration of methylation at the KvDMR1 or H19 loci and supports the notion that IGF2 overexpression is involved in the etiology of the tissue hypertrophy observed in different overgrowth disorders, including KTWS.

Expression of serogroup B meningococcal capsular polysaccharide is subject to frequent phase variation. A reversible +1/-1 frameshift mutation within a poly(dC) repeat altering the reading frame of the polysialyltransferase gene (siaD ), thereby causing premature arrest of translation, is responsible for loss of capsule expression. After analysis of transcription of the siaD gene from an encapsulated strain and from two unencapsulated derivatives, we have found that the siaD mRNA in the unencapsulated strains is reduced in size as a result of premature transcription termination at a cryptic Rho-dependent site within the proximal region of the siaD cistron. Termination is sensitive to bicyclomycin, a natural inhibitor of Rho activity. Bicyclomycin decreased the rates of capsule re-expression (off-on) without affecting the rates of loss of capsule expression (on-off). This finding suggested the existence of a novel mechanism linking transcription elongation termination and mutation frequency. A genetic system was therefore developed to measure phase variation of siaD-ermC' gene fusions in wild type and Rho-defective Escherichia coli strains. These studies demonstrated that in the Rho-defective E. coli strain readthrough transcription of the mutated siaD gene caused a fourfold lower off-on phase variation rate than in the congenic Rho+ strain. Analysis of phase variation of siaD-ermC' gene fusions in a DNA mismatch-defective E. coli strain suggests that the effect of transcription on mutation rates required a functional mismatch repair system.

Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria. Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in Escherichia coli by inhibiting the activity of transcription termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination region located within the hisG cistron of Salmonella typhimurium has been used as an experimental system. The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments. The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis. Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers.

The expression of Insulin-like Growth Factor 2 (IGF-2) and H19, two genes located on human chromosome 11p15 and provided with cell growth modulating activity, is regulated by parental imprinting, in that the activity of their alleles is dependent on the parental origin. Parental bias in the genetic alterations of chromosome 11p15 observed in several pediatric cancers suggests the involvement of imprinted genes in tumor development. We have previously reported that the number of functional IGF-2 alleles is frequently increased in rhabdomyosarcoma (RMS), as a consequence of either relaxation of imprinting (LOI) or gene duplication. Here we show that the expression of the H19 gene is significantly suppressed with respect to normal muscle tissue in 13 out of 15 rhabdomyosarcomas with embryonal histology (ERMS) and in three out of 11 rhabdomyosarcomas classified as alveolar subtype (ARMS). Since a growth-inhibitory activity has been found associated with the H19 gene, the extinction of its expression can contribute to RMS development. Parental imprinting of the H19 gene was found conserved in all informative RMSs, including those whose ICF-2 imprinting was relaxed, indicating that LOI is a gene-specific event. Seven ERMSs and one ARMS displaying low H19 RNA levels showed an underrepresentation of the expressed allele in their genotype. This result is consistent with the paternal imprinting of the H19 gene and with the preferential loss of the maternal 11p15 alleles in these neoplasms. Low H19 expression was also found in four out of eight RMSs retaining the heterozygosity at 11p15, but showing IGF-2 LOI. These findings suggest that the genetic and epigenetic alterations affecting chromosome 11p15 in a high number of RMSs cause deregulation of more than one imprinted gene, possibly affecting tumor growth, including the extinction of H19 expression and an increase in the number of active IGF-2 alleles.

Rab7 is a small GTPase localized to the late endosomal compartment. Its function was investigated by overexpressing dominant negative or constitutively active mutants in BHK-21 cells. The effects of such overexpression on the internalization and/or degradation of different endocytic markers and on the morphology of the late endosomal compartment were analyzed. We observed a marked inhibition of the degradation of 125I-low density lipoproteins in cells transfected with the Rab7 dominant negative mutants while the rate of internalization was not affected. Moreover in these cells there was an accumulation of many small vesicles scattered throughout the cytoplasm. In contrast, overexpression of the activating mutants led to the appearance of atypically large endocytic structures and caused a dramatic change in the distribution of the cation-independent mannose 6-phosphate receptor. Our data indicate that the Rab7 protein in mammalian cells is present on a late endosomal compartment much larger than the compartment labeled by the cation-independent mannose 6-phosphate receptor. Rab7 also appears to play a fundamental role in controlling late endocytic membrane traffic.

Increased prevalence of hepatitis C virus (HCV) infection has been found in patients with hepatocellular carcinoma (HCC). The expression of insulinlike growth factor II (IGF-II) has been linked to hepatocarcinogenesis in the experimental animal and in humans. Since reactivation of fetal IGF-II transcripts has been observed in human HCC, we have analyzed the levels of adult P1 and fetal P3 and P4 IGF-II promoter-derived transcripts in the liver of patients with HCV-related chronic active hepatitis (CAH), cirrhosis, and HCC by means of a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) assay. Transcripts derived from adult P1 promoter were increasingly expressed from normals to patients with CAH and cirrhosis, but were undetectable in the tumorous area of 5 of 7 HCC patients and present at low levels in the nontumorous area of all HCC patients. Transcripts derived from fetal P3 promoter were not detectable in normal subjects, while they were expressed abundantly in most CAH and all cirrhotic patients. Transcripts from fetal P4 promoter were detected at high levels in 3 of 9 CAH patients and in the majority of cirrhotic patients. Increased expression of fetal promoter-derived transcripts was also found in the liver of HCC patients, although levels were lower than in cirrhosis. Also, the activity of fetal P3 and P4 promoters was higher in the nontumorous than in the tumorous area of the liver of HCC patients. The expression of IGF-II transcripts was correlated with the rate of cell mitotic activity by measuring the expression of the proliferating cell nuclear antigen (PCNA) gene. PCNA messenger RNA (mRNA) levels progressively increased from normals to CAH and to cirrhotic patients, and persisted at a high level in the tumorous and in the nontumorous area of HCC subjects, thus showing that the increase of IGF-II transcripts in CAH and cirrhosis is accompanied by an activation of cell mitosis in these samples. These data suggest that the activation of IGF-II gent, expression from adult and fetal promoters may play a role in premalignant proliferation observed in HCV-related chronic liver disease.

Rab7 is a small GTP-ase localized on late endosomes, which regulates late endocytic membrane traffic in mammalian cells. Moreover it has been shown that this protein has a fundamental role in the cellular vacuolation induced by the cytotoxin VacA of Helicobacter pylori. We report here for the first time the isolation of a cDNA encoding human Rab7 from a placenta cDNA library. The open reading frame for human Rab7 encodes a protein of 207 amino acids which exhibits high homology with the mouse, rat, and dog counterparts. Northern blot analysis of total RNAs isolated from different cell lines with a cDNA probe containing the entire open reading frame revealed two mRNA transcripts of 2.5 and 1.8 kilobases. The isolation of human Rab7 cDNA will allow further characterization of its function in normal and pathological states.

A cDNA clone coding for the Rab7 protein was isolated from an NIH3T3 cell line (mouse fibroblasts) cDNA library. Sequence analysis shows high homology to the rat and dog cDNAs. Northern blot analysis showed the presence of two messenger RNA that differ at the 3' untranslated region.

Review. No abstract available.

Previous studies have shown that the expression of the his operon of Salmonella typhimurium is regulated at the level of transcription initiation, transcription elongation and RNA processing. We have analyzed his RNA in both prototrophic strains or strains harboring regulatory and auxotrophic mutations grown under a variety of metabolic conditions that lead to differential expression of the operon. Under some of these conditions, there is an increase in the amount of prematurely released his-specific RNA, resulting in modulation of the relative amount of full-length transcripts. Under the same metabolic conditions, there is also a modulation of RNA processing events that generate a very stable RNA species comprising the five distal cistrons. These effects appear to be due to perturbation of the translation process caused by alterations in the intracellular pool of initiator transfer RNA.

We have cloned and sequenced the genomic regions encompassing the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium. Rho factor of S. typhimurium has only three amino acid differences with respect to the Escherichia coli homolog. Northern (RNA) blots and primer extension experiments were used to characterize the N. gonorrhoeae rho transcript and to identify the transcription initiation and termination elements of this cistron. The function of the Rho factor of N. gonorrhoeae was investigated by complementation assays of rho mutants of E. coli and S. typhimurium and by in vivo transcription assays in polar mutants of S. typhimurium.

We have constructed an expression vector carrying the Escherichia coli his operon control region to study the ability of defined segments of DNA to cause rho factor-mediated transcription termination both in vivo and in vitro. We have previously identified a consensus motif consisting of a region of high cytosine over guanosine content common to several cryptic intracistronic transcription termination elements unmasked by polar mutations. We show that a DNA fragment possessing features similar to the ones previously identified is capable of causing rho-mediated mediated release of transcripts in vivo and in vitro. The efficiency of termination depends on the length and efficiency of termination depends on the length and relative cytosine over guanosine ratio of the element.

We have characterized at the molecular level several polar mutations in four different cistrons of the his operon of S. typhimurium. An analysis of the his-specific transcripts produced in vivo in the mutant strains, together with in vitro transcription assays, led to the identification of several cryptic Rho-dependent transcription termination elements within the his operon that are activated by the uncoupling of transcription and translation. Common features of these elements were sought and found with a computer program. We have identified a consensus motif, consisting of a cytosine-rich and guanosine-poor region, that is located upstream of the heterogeneous 3' endpoints of the prematurely terminated in vivo transcripts and that is present in all the Rho-dependent transcription terminators described thus far.

The rat insulin-like growth factor II (rIGF-II) gene, which exists as a single copy in the genome, is expressed as a multitranscript family of mRNA molecules ranging in size from 4.6 to 1 kilobases. Part of this heterogeneity can be ascribed to the presence of two different promoters, each transcribing alternative 5'-noncoding regions which are spliced to common coding exons. In the present study we use a combination of DNA sequence analysis of the gene, mapping of the mRNA molecules by Northern analysis and ribonuclease protection experiments, and DNA sequence analysis of cDNA clones complementary to different regions of the genome to establish the structure of several rIGF-II mRNA species. These results indicate that RNA heterogeneity also arises from the use of different polyadenylation sites. In addition, a variant 2 kilobases RNA was observed that was colinear with the distal 1700 base pairs of the 3147 base pair long exon 3, and may arise by alternative RNA splicing. These posttranscriptional modifications of RNAs arising from the rIGF-II transcription unit may generate molecules with different functional potential.

The entire histidine operon of Escherichia coli K-12 was cloned in the vector plasmid pBR313, and a complete restriction map of the operon was determined. By using subclones, complementation tests, and enzyme assays, we were able to make a correlation between the physical map and the genetic map of the operon. We determined the sequence of a fragment of DNA 665 base pairs long, comprising the distal portion of the hisC gene, the proximal portion of the hisB gene, and the internal transcription initiation site hisBp. The efficiency of this promoter was assessed under different physiological conditions by cloning the DNA fragment in a recombinant vector system used to study transcriptional regulatory signals. The precise point at which transcription initiates was determined by S1 nuclease mapping.

Several permanent cell lines of epithelial origin are able to grow in culture in the absence of serum components. It was originally proposed by Temin et al. (1972) that such behaviour was dependent on the production of mitogenic factors (MSA or Multiplication Stimulating Activity) . The most studied system is a rat liver cell line BRL 3A isolated by Coon (1968) . Subsequent studies demonstrated that MSA is the rat equivalent of the insulin like growth factor II (IGF-II) (Rechler et al., 1985), but also showed that the ability of this cell line to grow in serum-free medium is independent of the synthesis and secretion of this polypeptide (Nissley et al., 1977) . We have been investigating this system mainly to understand the biological role and the expression and regulation of the IGF-II gene (Chiariotti et al., 1988). In the course of screening several cDNA libraries derived from the BRL 3A cell line we accidentally isolated some cDNA clones abundantly represented in the stable mRNA population, but not related to IGF-II. Subsequent characterization of these clones lead to the discovery that this mRNA code for a protein of molecular weight 22,800 belonging to the superfamily of ras-related genes (Bucci et al., 1988). In the present study we report some aspects of the organization, structure and expression of this novel putative GTP-binding protein.

We have determined the complete nucleotide sequence of the histidine operons of Escherichia coli and of Salmonella typhimurium. This structural information enabled us to investigate the expression and organization of the histidine operon. The proteins coded by each of the putative histidine cistrons were identified by subcloning appropriate DNA fragments and by analyzing the polypeptides synthesized in minicells. A structural comparison of the gene products was performed. The histidine messenger RNA molecules produced in vivo and the internal transcription initiation sites were identified by Northern blot analysis and S1 nuclease mapping. A comparative analysis of the different transcriptional and translational control elements within the two operons reveals a remarkable preservation for most of them except for the intercistronic region between the first (hisG) and second (hisD) structural genes and for the rho-independent terminator of transcription at the end of the operon. Overall, the operon structure is very compact and its expression appears to be regulated at several levels.

In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the his IE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, L-histidinol:NAD+ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxyl-terminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps.